SCT60103 GTC Group 1 Presentation Strategies are focussed more in cancer treatment rather than prevention

1. Therapeutic strategies that target the
cellular transformation process for cancer
prevention and treatment.
By Chong Jia En, Chong Jia Yii, Lim Yee Hung, Tan Liu Xi and Yeap Tze Huay
SCT60103 Genes and Tissue Culture Technology

2. Tumour Development
 Uncontrolled growth
 Abnormal chromosomes
 No apoptosis
 No contact inhibition
 Ability to metastasise
 Non specialised cells
 Immature / undifferentiated
 Variable appearance
 Variable shaped nuclei
 Disorganised arrangement

3. • The result of a series of events that both depends on and promotes
instability.
• Alters many of the cell line’s properties:
• Can arise from
Transformation
 Growth rate
 Mode of growth
 Specialised product formation
 Longevity
 Tumourigenicity
 Infection with virus
 Transfection with genes
 Ionising radiation
 Treatment with chemical carcinogens (Freshney 2016, p.495)
(Fearon and Vogelstein 1990).

4. WNT Signalling Pathways
WNTs: secreted glycoproteins (Papkoff, Brown and Varmus 1987) which regulate multiple signalling
pathways via both β-catenin (CTNNB1)- dependent and CTNNB1-independent mechanisms
WNT-CTNNB1 signalling (maintenance of tumour-initiating cells) and CTNNB1-independent WNT
signalling pathways can either activate or inhibit tumorigenesis and cancer progression in a
context-dependent manner
Can promote transcriptional changes that can drive epithelial-mesenchymal transition – alteration in protein
expression that results in complex changes in cell behaviours (e.g. cell-cell interaction and enhanced motility)
Changes in WNT signalling pathways and other oncogene and tumour suppressor pathways
cooperate to drive cancer initiation and progression
Targeted cellular pathways/targets:
• WNT signalling pathways
• specific overexpressed WNTs and WNT receptors in tumours (WNT receptors retain the
malignant phenotypes but are not necessary for normal tissue homeostasis)
How does it lead to cellular transformation?

5. Therapeutic strategies:
• Small molecules
• lithium chloride- cause CTNNB1 activation by inhibiting GSK3
• IWP (inhibitor of WNT production)- a membrane bound acetyltransferase that modifies
WNT ligands for their secretion and signalling activity (Chen et al 2009).
• DVL-specific inhibitors – to inhibit DVL function in CTNNB1-dependent and CTNNB1-
independent pathways by protein-protein interaction and structure-based design
algorithms (Fujii et al 2007).
• Blocking antibodies (target specific overexpressed WNTs and WNT receptors in tumours
that inhibit proliferation and cause apoptosis in different cancers (He et al 2004)).
• FZD7-specific antibodies that targets FZD receptors in hepatocellular carcinoma cells
blocks expression of WNT-CTNNB1 gene (Wei, Chua, Gapper and So 2011) and interacts with
DVL to inhibit the growth of HCC cells (Nambotin et al 2011).
WNT Signalling Pathways

6. Therapeutic strategies:
WNT Signalling Pathways
• Peptides
• FZD7-blocking peptides- block TCF/LEF reporter activity and the expression of WNT-
CTNNB1 target genes in HCC cells; disrupt the growth of HCC cells (did not affect the
viability of normal hepatocytes lacking FDZ7 expression) -> induce apoptosis in colon
and breast cancer cells
• Use of peptide ligands that binds to the PDZ domain of FZD2 to disrupt WNT-CTNNB1
signalling
• WNT signalling and combination therapy (sensitize or desensitize cancer cells to
toxic insults)
• Cancer cells’ sensitivity to chemotherapeutic agents is enhanced by inhibiting or activating
(overexpression of WNT5A) the WNT-CTNNB1 pathway> cooperative inhibition of tumour
growth
• Gene therapy
• FZD7 knockdown – reduces TCF-dependent transcription and xenograft tumour growth
of triple negative breast cancer
• Small interfering RNAs (siRNA) – to reduce ROR1 expression as is decreases the growth
of gastric, lung and breast cancer cells during cell culture and xenografts / induce
apoptosis in CLL, breast carcinoma and cervical carcinoma

7. WNT Signalling Pathways
Challenge
 Mutations in APC and AXIN1 hyperactivates the
WNT-CTNNB1 pathway, limiting the potential
molecular targets for pathway modulation. It is
because factors acting upstream of the
destruction complex are not required in
pathway activation.
Solution
 Screening identifies molecules which affect the
interaction between TCF7L2 and CTNNB1 and
hence, inhibit CTNNB1-dependent transcription.
• WNT signalling cannot be targeted using a single
universal strategy as the activation / inhibition of
WNT signalling and WNT-CTNNB1 independent
signalling pathways in cancer progression are in a
context-dependent manner / signalling cascade
regulation is dependent on the combination of
receptors that are expressed only by a particular
cancer type
------------------- -------------------
Current development:
 further investigation on the mechanisms of crosstalk between WNT pathways and related signalling
networks such as oncogene and tumour suppressor pathways
 combinatorial therapies
 identify the genetic factors and biomarkers that allow prediction on responses to treatment with
WNT pathway modulators
 identify WNT receptors that are necessary for cell evasion of senescence and apoptosis
------------------------------------------

8. Therapeutic Strategy: Metformin
---------------------------------------------------------------------
Treat Type II Diabetes Mellitus by inhibiting lipogenesis and mitigates hyperlipidaemia, reduce cellular levels of reactive
oxygen species and down regulates proinflammatory cytokines which constitutes risk factor of cancer
Association between Diabetes Mellitus and Cancer
• First described 81 years ago by Joslin clinician
• Risk increase in diabetes
• Highest for liver, pancreas and endometrial cancer
• Secondary for colorectal, breast and bladder cancer
• No association between diabetes and lung cancer
• Inverse association with prostate cancer were found
Early 1980s, Dilman et al. gave phenformin to patients with breast and colon cancer to correct dysregulated metabolism and they
found that both primary and metastatic tumours were suppressed.
How a diabetes patient’s cell transform into tumour cell?
Function as – anti-tumour effect
• Depress tumour proliferation (AMPK)
• Induce apoptosis, autography, cell cycle arrest of
tumour cells
• Reduce DMBA-induced tumour by 80% and inhibits
tumorigenesis of mammary gland and delay onset of
tumours
• Insulin resistance
• Caused by the increased level of insulin and insulin-like growth factor (I/IGF)
• This bind to some receptor of cell
• Activate the downstream signalling pathway
• Development of tumour
• During development of tumour, may produce cytokines and tumour necrosis factor - alpha (TNF - ⍺), interleukins (IL-6) and etc
• This may activate the nuclear factor k-B and signal transducer to activate the transcription of tumour cells

9. Metformin
EPIDEMIOLOGICAL EVIDENCE OF ANTI-TUMOR EFFECT OF
METFORMIN
Associations between metformin use and reduction of cancer risk obtained mostly from T2DM patients
• Findings
• Reduced risk of cancer
• Improves overall survival
• Suggesting metformin exerts therapeutic effect
• First
• Case control study reviewed clinical records from 923 T2DM patients in UK and found 23% reduction in risk of
developing cancer (Evans et al. 2005)
• Since then, many studies further looked into association between metformin use and the risk of site-specific cancers
• Meta-analysing using 18 observational studies and 561,836 patients revealed that metformin use associated with
overall 27% reduction in risk of developing all types of cancer
• Strong association between metformin use and reduced risk found in cancers for liver, colorectal, pancreatic,
stomach and oesophageal cancers, whereas no consistent results found on cancers from breast, prostate and lung
-----------------------------------------------------------------------------

10. AMPK Pathway
Metformin will inhibit proliferation of breast cancer cell through activation of AMPK
and inhibition of mTOR (AMPK-dependent which is block by small interfering RNA
against AMPK)
1. AMPK directly phosphorylate tuberous sclerosis complex 2(TSC2)
2. Activate TSC1/TSC2 compound inhibit activity of Ras homolog
3. mTOR
4. AMPK directly phosphorylates the mTOR binding partner receptor
5. Inactivate receptor and mTOR
Metformin may activate expression of AMPK, inhibit raptor and mTOR and its downstream
ribosome S6 protein kinase (p70S6K)
Metformin
Function of this pathway
• Promote cell mitosis, stimulate cell growth,
inhibit cell apoptosis
• Reduce blood insulin level which inactivate
I/IGF signalling pathway, improved insulin
resistance (Goodwin et al. 2008)
• Directly inhibit mTOR by activating AMPK
(liver tissue) or indirectly inhibit mTOR by
decrease activation of insulin receptor / IGF
- 1 receptor and Akt in lung tissue
I/IGF pathway
1. Metformin reduce the level of
I/IGF - 1 in blood circulation
2. Inactivate the downstream
P13K/Akt/mTOR signalling
pathway
3. Inhibit tumour cell

11. Challenges
DOSE
• Metformin prosecutes anticancer function via systemic but indirect
effect
• Mediated by improving hyperglycaemia and hyperinsulinemia
• Directly acts on cancer cells by inhibiting growth-promoting factors or
activating tumour suppressors
• Occurs through AMPK-dependent and independent mechanisms
• Concentration required for direct effect of metformin
• within the range of 5 to 10 mM which is greater than the steady state levels
of plasma when standard dose is given to T2DM patients
• Low concentration of metformin is not sufficient to cause AMPK
activation
• insufficient to inhibit malignant growth of cancer cells
Future Possible Solution
SITE
• According to studies in mice, administration of metformin at 50mg/kg
per day results in maximal concentration of 50 to 60 µM in hepatic
portal vein
• Greatest accumulation occurs in the small intestine and secondly in
stomach, colon, kidney and liver
• This suggest that tumour originated from these sites could be the first
target for orally administered metformin
Metformin
-------------------
-----------------------------
DOSE
Addition of other activators with different mechanisms may circumvent the limitation
low doses
• E.g.: salicylate binds to a site different from AMP and directly activates AMPK
• This suggest combined use of metformin and salicylate may generate synergistic
effect which complement the limitation of Metformin
SITE
Suggest other route of administration
• E.g.: Intravenous or targeted therapy

12. References
Chen, B, Dodge, ME, Tang, W, Lu, J, Ma, Z, Fan, C, Wei, S, Hao, W, Kilgore, J, Williams, NS, Roth, MG,
Amatruda, JF, Chen, C and Lum, L 2009, ‘Small molecule-mediated disruption of Wnt-dependent signalling in
tissue regeneration and cancer’, Nature Chemical Biology, vol. 5, no. 2, pp. 100-107.
Dilman, VM, Berstein, LM, Ostroumova, MN, Fedorov, SN, Poroshina, TE, Tsyrlina, EV, Buslaeva,
VP, Semiglazov, VF, Seleznev, IK, Bobrov, YF, Vasilyeva, IA, Kondratjev, VB, Nemirovsky, VS and Nikiforov, YF
1982, ‘Metabolic immunodepression and metabolic immunotherapy: an attempt of improvement in immunologic
response in breast cancer patients by correction of metabolic disturbances’, Oncology, vol. 39, pp. 13–19.
Dilman, VM, Berstein, LM, Yevtushenko, TP, Tsyrlina, YN, Ostroumova, MN, Bobrov, YF, Revskoy, YS,
Kovalenko, IG and Simonov, NN 1988, ‘Preliminary evidence on metabolic rehabilitation of cancer patients’, Arch
Geschwulstforsch, vol. 58, pp. 175–183.
Evans, J, Donnelly, L, Emslie-Smith, A, Alessi, D and Morris, A 2005, Metformin and reduced risk of cancer in
diabetic patients. BMJ, vol. 330, pp. 1304–1305.
Fearon, ER and Vogelstein, B 1990, ‘A geneteic model for colorectal tumorigenesis’, Cell, vol. 61, pp. 759-767.
Franciosi, M, Lucisano, G, Lapice, E, Strippoli, GF, Pellegrini, F and Nicolucci, A 2013, ‘Metformin therapy and
risk of cancer in patients with type 2 diabetes: systematic review’, PLoS One, vol. 8, e71583.
Freshney RI 2016, Culture of Animal Cells – A Manual of Basic Technique and Specialised Applications, 7th
edition, John Wiley & Sons, Inc., Hoboken, New Jersey.
Fujii, N, You, L, Xu, Z, Uematsu, K, Shan, J, He, B, Mikami, I, Edmondson, LR, Neale, G, Zheng, J, Guy, RK and
Jablons, DM 2007, ‘An antagonist of dishevelled protein-protein interaction suppresses β-catenin-dependent
tumor cell growth’, Cancer Research, vol. 67, no. 5, pp. 573-579.
Goodwin, PJ, Pritchard, KI, Ennis, M, Clemons, M, Graham, M and Fantus, IG 2008, ‘Insulin-lowering effects of
metformin in women with early breast cancer’, Clin Breast Cancer, vol 8, pp. 501-505.
He, B, You, L, Uematsu, K, Xu, Z, Lee, AY, Matsangou, M, McCormick, F and Jablons, DM 2004, ‘A monoclonal
antibody against Wnt-1 induces apoptosis in human cancer cells, Neoplasia, vol. 6, no. 1, pp. 7-14.
He, H, Ke, R, Lin, H, Ying, Y, Liu, D and Luo, Z 2015, ‘Metformin, an old drug, brings a new era to cancer
therapy’, Cancer J, vol. 21, no. 2, pp. 70-74.
Hudecek, M., Schmitt, T.M., Baskar, S., Lupo-Stanghellini, M.T., Nishida, T., Yamamoto, T.N., Bleakley, M.,
Turtle, C.J., Chang, W.C., Greisman, H.A., Wood, B., Maloney, D.G., Jenson, M.C., Rader, C. and Riddell, S.R.
2010, ‘The B cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-
specific chimeric antigen receptor’, Blood, vol. 116, no. 22, pp. 4532-4541.
Kasznicki, J, Sliwinska, A and Drzewoski, J 2014, ‘Metformin in cancer prevention and therapy’, Annals of
Translational Medicine, vol. 2, no. 6, pp 57-67.
Nambotin, SB, Lefrancois, L, Sainsily, X, Berthillon, P, Kim, M, Wands, JR, Chevallier, M, Jalinot, P, Scoazec, JY,
Trepo, C, Zoulim, F and Merle, P 2011, ‘Pharmacological inhibition of Frizzed-7 displays anti-tumor properties in
hepatocellular carcinoma, Journal of Hepatology, vol. 54, no. 2, pp. 288-299.
Papkoff, J, Bown, AM and Varmus, HE 1987, ‘The int-1 proto-oncogene products are glycoproteins that appear to
enter the secretory pathway’, Molecular Cellular Biology, vol 7, no. 11, pp. 3978-3984.
Wei, W, Chua, MS, Grepper, S and So, SK 2011, ‘Soluble Frizzled-7 receptor inhibits Wnt signalling and
sensitizes hepatocellular carcinoma cells towards doxorubicin, Molecular Cancer, vol. 10, no. 16, p. 16.
Wu, A, Oh, S, Wiesner, SM, Ericson, K, Chen, L, Hall, WA, Champoux, PE, Low, WC and Ohlfest, JR 2008,
‘Persistence of CD133+ cells in human and mouse glioma cell lines: detailed characterization of GL261 glioma
cells with cancer stem cell-like properties’, Stem Cells and Development, vol. 17, no. 1, pp. 173-184.
Yang, J, Baskar, S, Kwong, KY, Kennedy, MG, Wiestner, A and Rader, C 2011, ‘Therapeutic potential and
challenges of targeting receptor tyrosin kinase ROR1 with monoclonal antibodies in B-cell malignancies’, PLoS
ONE, vol. 6, no. 6, e21018.
Zhang, P, Li, H, Tan, X, Chen, L and Wang, S 2013, ‘Association of metformin use with cancer incidence and
mortality: a meta-analysis’, Cancer Epidemiol, vol. 37, pp. 207–218.
Zi, FM, Zi, HP, He, JS, Shi, QZ and Cai, Z 2018, ‘Metformin and cancer : an existing drug for cancer prevention
and therapy (Review)’, Oncology Letters, vol. 15, pp. 683-690.